The present invention is directed to new and useful processes for the preparation of 2′-O-substituted purine nucleosides. 2′-O-Substituted purine nucleosides are important compounds used routinely for the synthesis of oligonucleotides and related compounds.
Currently, 2′-O-methoxyethylpurine is prepared from the conversion of a purine into 2,6-diaminopurine riboside using corrosive triflic acid and heating at 150° C. under pressure for several days, followed by the alkylation with methoxyethyl bromide to yield 2,6-diaminopurine riboside, and the conversion back to the desired alkylated 2′-O-methoxyethylpurine using the adenosine deaminase enzyme. However, the alkylation step produces a mixture of various substituted alkylated products including mono-substituted 2′-O-methoxyethyl-2,6-diaminopurine riboside, 3′-O-methoxyethyl-2,6-diaminopurine riboside, and di-substituted 2′-O-3′-O-dimethoxyethyl-2,6-diaminopurine riboside. Furthermore, methoxytheyl bromide is unstable and its CH3—O bond is easily cleaved with traces amount of HBr to generate methyl bromide, which leads to a more reactive electrophile and therefore forming 2′-O-methoxyl-2,6-diaminopurine riboside that is very difficult to be separated. Lastly, the conversion of the already low yielding 2′-O-methoxyl-2,6-diaminopurine riboside requires the use of a relatively expensive adenosine deaminase enzyme to accord the desired 2′-O-methoxyethyl purine in an unattractive overall yield. Therefore, there is a need in the art to discover a more efficient and less expensive method to synthesize 2′-O-methoxyethylpurines.
Oligonucleotides and their analogs have been developed for various uses in molecular biology, including use as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels such as fluorescein, biotin, digoxigenin, alkaline phosphatase or other reporter molecules. Modifications also have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. These modifications include use of methyl phosphonates, phosphorothioates, phosphorodithioate linkages, and 2′-O-methyl ribose sugar units. Other modifications have been directed to the modulation of oligonucleotide uptake and cellular distribution. The success of these oligonucleotides for both diagnostic and therapeutic uses has created an ongoing demand for improved oligonucleotide analogs.
Oligonucleotides can be synthesized to have custom properties that are tailored for a desired use. Thus a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents and as therapeutic entities.